Ghiafeh Davoodi, F., Javan, M., Ahmadiani, A. (2010). The Effect of Swim Stress on Morphine Tolerance Development and the Possible Role of Nitric Oxide in this Process. Iranian Journal of Pharmaceutical Research, Volume 4(Number 3), 167-173.

F Ghiafeh Davoodi; M Javan; A Ahmadiani. "The Effect of Swim Stress on Morphine Tolerance Development and the Possible Role of Nitric Oxide in this Process". Iranian Journal of Pharmaceutical Research, Volume 4, Number 3, 2010, 167-173.

The Effect of Swim Stress on Morphine Tolerance Development and the Possible Role of Nitric Oxide in this Process

It has been shown that stress and chronic pain could prevent the development of tolerance to morphine analgesia, which appears to be related to the activation of hypothalamus–pitutitary–adrenal (HPA) axis, activation of neuroendocrine systems and changes in neurochemical levels. Moreover, the involvement of nitric oxide (NO) in the development of tolerance to morphine analgesia has been implicated. In the present study, we have tried to investigate the effect of swim stress, as a painless kind of stress, on the development of tolerance to find out whether the inhibition of tolerance is mediated by the direct effect of pain on the pain conduction pathway, or by its stress aspect. Besides, we evaluated the probable interactions between swim stress, nitric oxide level and the development of morphine tolerance. Adult male Wistar rats, weighing 180-220 g, were used in all these experiments. The experimental groups received chronic morphine (20 mg/kg, i.p), swim stress in 20?C water bath (4 min), or a combination of swim stress and chronic morphine (20 mg/kg, i.p), each for 4 days, while the first control group received saline (1 ml/kg, i.p) for 4 days. On the 5th day, all the experimental and control groups received a single dose of morphine (10 mg/kg i.p). The second control group received saline for 5 days. The intact group received only one single dose of morphine (10 mg/kg, i.p). All the mentioned groups were subjected to tail-flick and formalin tests on the 5th day. Other experimental groups were subjected to the assay for measuring nitrite as an indicator of NO, using the Griess method. Our results showed that co-administration of swim stress with chronic morphine prevented the development of morphine tolerance and the level of NO increased in the presence of swim stress (p<0001). The combination of morphine and swim stress significantly decreased NO production in comparison with the chronic morphine administered group (p<0.001). These data suggest that the activation of HPA axis and consequently the suppression of (NO) production induced by chronic morphine, lead to the inhibition of morphine tolerance.

It has been shown
that stress and chronic pain could prevent the development of tolerance to
morphine analgesia, which appears to be related to the activation of
hypothalamus?pitutitary?adrenal (HPA) axis, activation of neuroendocrine
systems and changes in neurochemical levels. Moreover, the involvement of
nitric oxide (NO) in the development of tolerance to morphine analgesia has
been implicated. In the present study, we have tried to investigate the effect
of swim stress, as a painless kind of stress, on the development of tolerance
to find out whether the inhibition of tolerance is mediated by the direct
effect of pain on the pain conduction pathway, or by its stress aspect.
Besides, we evaluated the probable interactions between swim stress, nitric
oxide level and the development of morphine tolerance. Adult male Wistar rats,
weighing 180-220 g, were used in all these experiments. The experimental groups
received chronic morphine (20 mg/kg, i.p), swim stress in 20?C water bath (4
min), or a combination of swim stress and chronic morphine (20 mg/kg, i.p),
each for 4 days, while the first control group received saline (1 ml/kg, i.p)
for 4 days. On the 5th day, all the experimental and control groups
received a single dose of morphine (10 mg/kg i.p). The second control group
received saline for 5 days. The intact group received only one single dose of
morphine (10 mg/kg, i.p). All the mentioned groups were subjected to tail-flick
and formalin tests on the 5th day. Other experimental groups were
subjected to the assay for measuring nitrite as an indicator of NO, using the
Griess method. Our results showed that co-administration of swim stress with
chronic morphine prevented the development of morphine tolerance and the level
of NO increased in the presence of swim stress (p<0001). The combination of
morphine and swim stress significantly decreased NO production in comparison
with the chronic morphine administered group (p<0.001). These data suggest
that the activation of HPA axis and consequently the suppression of (NO)
production induced by chronic morphine, lead to the inhibition of morphine
tolerance.

Keywords: Morphine; Swim stress; Tolerance; Nitric oxide;
Analgesia.

Introduction

It has been reported that chronic opioid treatment could lead to the
development of antinociceptive tolerance in both human and laboratory animals
(1), which is one of the major problems of using morphine as an analgesic drug.
Clinical studies, however, have indicated that tolerance and dependence are not
a major concern when opiates are used to control pain (2, 3). For instance,
chronic morphine administered in the presence of formalin-induced pain does not
lead to the development of analgesic tolerance in rats (3- 6).

There are some evidence indicating that the
hypothalamic-pituitary-adrenal (HPA) axis is involved in the development of
tolerance to morphine analgesia and could respond to stressful stimuli such as
formalin induced pain (7-9). For example, it has been found that
adrenocorticotropin hormone can prevent the development of tolerance to
morphine analgesia (10) or stress produced by the same effect on intact mice,
but not on the adrenalectomized mice (11). Both adrenalectomy (12) and
hypophysectomy have been shown to potentiate the opiate tolerance and
interestingly, the effects of hypophysectomy were reversed by the replacement
of ACTH (13). These results raise the possibility that blockage of tolerance development
by formalin-induced pain is related to the activation of HPA axis, following
formalin injection. Supportive data by Vacarrino and Couret (3) showed that
genetical differences in stress induced HPA activity, may contribute to
differential development of tolerance to morphine analgesia during pain. The
inhibition of tolerance development by formalin-induced pain depends on
corticosterone activity, such that an increase in corticosterone triggered by
the stress fullness of pain, acts to attenuate the tolerance development (14).
Moreover, it has been demonstrated that chronic administration of opioids leads
to the development of tolerance to their stimulatory effects on the HPA axis
(15-17). The effect of opioids on the HPA axis is currently thought to be
mediated, directly or indirectly, by the release of corticotropine releasing
factor (CRF) (18).

Recent studies have suggested that opioid tolerance may also be mediated
by increased production of nitric oxide (NO). NO has also been implicated in
nociception processing (19). It has been shown that nitric oxide synthase (NOS)
inhibitors attenuate tolerance development following chronic morphine
administration (20-23). It has also been reported that increased NO production
potentiates morphine analgesia and enhances the development of morphine
tolerance in mice (24). NO production is one of the mechanisms that has been
shown to be involved in the development and maintenance of morphine tolerance,
in addition to the activation of NMDA receptors, translocation and activation
of protein kinase C (PKC( (25).

The localization of constitutive NOS (cNOS) in hypothalamic cells, which
regulates the activity of the HPA axis, suggests that NO may play a
physiological role in HPA axis activation following different stimuli. The role
of endogenous NO in the regulation of the HPA axis activity remains
controversial, because in different preparations contradictory results have
been reported. Both stimulatory and inhibitory effects of NO on the stimulated
release of CRH were observed in a short-term -culture of the hypothalamus
tissue (26). On the other hand, stress induced NO production in the adrenal
cortex, attenuates the adrenal corticosterone release stimulated by
stress-induced ACTH release, or facilitates recovering of the increased
corticosterone to the basal level (27). In support of this hypothesis,
Pasternak et al. (28) reported the implication of NO in the development of
tolerance and dependence to mu receptor agonists. They demonstrated that a NOS
inhibitor blocked the development of tolerance to morphine (28, 29). NO may
modulate the release of corticosterone from the adrenal cortex in-vivo
and it has been found that immobilization induced stress, provoke a marked
increase in neuronal NOS (nNOS) mRNA expression in the adrenal cortex (27, 30).

Therefore, considering the inhibitory effect of chronic pain on the
development of tolerance to opioids, which is accounted by the stress aspect of
pain and activation of HPA axis, the present study was designed to study the effect
of swim stress, as a non painful stress, on the development of tolerance. In
parallel, we also measured the amount of nitrite, as a metabolite of NO, to
investigate the probable correlation between NO production and swimstress
induced inhibition of morphine tolerance.

The swim stress
test was carried out in a cylindrical plastic container, 28 cm in diameter and
44cm in height. The level of water ranged between 30-35 cm above the floor, so
that in all cases escape from the cylinder was impossible. The water temperature
was monitored carefully and maintained at 20?1?C To examine the effect of swim
stress on the development of tolerance to morphine analgesia, rats were
individually made to swim daily for 4 min in water for 4 days (31).

Morphine Tolerance induction

Rats were rendered tolerant by daily injections of morphine (20 mg/kg),
dissolved in physiological saline and administered intrapritoneally (i.p) for 4
days (3).

Assessment of morphine analgesia

Twenty-four hours after the final
injection for tolerance induction, pain sensivity was assesed using the
tail-flick and formalin tests as described previously (32), after
administration of a single dose of morphine (10 mg/kg, i.p) on the 5th
day.

Measurment of nitrite

Animals were decapitated to collect
their blood. The serum samples were separated and stored at -70?C until the
measuring time. NO was measured using Griess reaction (33, 34).

Test groups

Saline: These animals received saline for 5 days. Sal/M: These animals
received saline for 4 days and a single dose of morphine (10 mg/kg, i.p) on the
5th day. M: These animals received morphine for 4 days (20
mg/kg,i.p) and a single dose of morphine (10 mg/kg, i.p) on the 5th
day. SS/M: These animals received swim stress for 4 days and a single dose of
morphine (10 mg/kg, i.p) on the 5th day. SSM/M: These animals
received swim stress combined with morphine (20mg/kg) for 4 days and a single
dose of morphine (10 mg/kg, i.p) on the 5th day. Intact: These
animals received only a single dose of morphine (10 mg/kg, i.p) on the 5th
day.

Statistical analysis

All the data were analyzed using paired
and unpaired t-tests and the one?way analysis of variance (ANOVA), followed by
Tukey?s test for multiple comparison. P<0.05 was considered as significant.

Results and Discussion

As shown in Figures 1 and 2, a
significant tolerance to morphine (10 mg/kg, i.p) was induced in rats receiving
chronic morphine (20 mg/kg,i.p for 4 days) in the absence of stress, but not in
the rats receiving the similar doses of morphine in the presence of swim stress
(p<0.05). There was no significant difference between the animals that
received a single dose of morphine (10 mg/kg) on the 5th day (intact
rats) and those which received swim stress for 4 days and a single dose of
morphine on the 5th day.

The results of the formalin test
indicated that in acute phase, the rats that received swim stress and those
which received chronic morphine combined with the swim stress, both showed
significant analgesia comparing to the animals that received saline or chronic
morphine (p<0.01) (Fig. 3).

In chronic phase of the formalin test, the animals which
received only swim stress or the combination of swim stress and chronic
morphine, showed significant decrease in the development of tolerance in
comparison with those that received chronic morphine (p<0.01 and p<0.05,
respectively) (Fig.4).

Our results showed that NO production increased significantly in
the animals that received chronic morphine, compared with the control group
(p<0.001). Moreover, NO production increased in the rats that received swim
stress and those that received a combination of chronic morphine and swim
stress, but this increase was significantly lower than that of the animals which
received only chronic morphine (p<0.01, Fig.5).

It has been reported that chronic pain inhibits the development of
morphine tolerance and this effect is mediated through the stress aspect of
pain and the activation of HPA axis. Chronic morphine administration can
suppress HPA axis and leads to the development of tolerance. It should also be
noted that stress could activate the opioid system (14). The inhibitory effect
of foot shock and psychological stresses on the tolerance development has been
demonstrated to be mediated through the Vasopersin?Arginine system (35).
Stress, through adrenal glucocorticoids and ACTH, can prevent the development
of tolerance to morphine analgesia (10, 11) and adrenalectomy or hypophysectomy
enhances the development of analgesic tolerance (12, 13). The effects of hypophysectomy
could be eliminated by the administration of ACTH (13). Therefore, it is
possible that stress induced HPA activity contributes to the inhibitory effect
of pain on the development of morphine tolerance (3). Finally, it has been
shown that low doses of β?endorphin and morphine
increase the secretion of corticosterone, while higher concentrations of
opiates (e.g. the chronic administration of morphine) decreases it (36, 37).
The inhibitory effect of morphine on the corticosterone secretion in pups
exposed to chronic morphine reflects both decreased release of ACTH and direct
inhibition of steroid synthesis caused by the chronic morphine regimen
(38).

Our results showed that swim stress can attenuate the development of
morphine tolerance in both tail flick (Fig. 1 and 2) and formalin tests (Fig. 3
and 4).

Treatment of rats with swim stress and the combination of chronic
morphine and swim stress resulted in an increased NO production, which was
significantly lower than that produced in the animals that received only
chronic morphine (Fig. 5). These results indicate that swim stress could
attenuate NO production probably through activation of HPA axis, which
consequently results in the increased secretion of ACTH. It has also been
reported that the activation of ATP sensitive potassium channels following NO
production participates in development of tolerance to morphine analgesia (39).
NO inhibitors could prevent the development of morphine tolerance (40) and
administration of N-methyl?D-aspartate receptor antagonist and NO synthesis
inhibitors, can suppress the development of morphine tolerance. Moreover, in
the spinal cord of the rats rendered tolerant to morphine, the expression of
nNOS increases (41). These data suggest that NO plays a role in the development
of morphine tolerance. Bugajski et al. (26) suggested that NO may play a
physiological role in the response of HPA axis to different stimuli. The role
of endogenous NO in regulating the activity of the HPA axis remains
controversial, as contradictory results have been reported in different
preparations.

Blockage of NO synthesis significantly impaireds the release of ACTH in
response to a mild electroshock and water avoidance stress, which causes rapid
activation of the HPA axis (26). There are some reports that the inhibition of
NOS can potentiate continuous cold water swimming (CCWS) antinociception, which
indicates that the inhibition of NOS could probably affect a selective form of
pain inhibition (19). N-nitro-L- arginine (L-NA) or its methyl ester (L-NAME)
could potentiate antinociception elicited by the continuous cold water swimming
(19, 42).

NO antagonists have been reported to reduce the development of morphine
antinociceptive tolerance (22, 23). Hence, the effects of swim stress on
morphine tolerance are probably the result of the inhibition of NO production.
Although a moderate increase in NO production was observed following the swim
stress, there was no change in morphine analgesia in this group of animals. As a
result, it seems that a moderate increase in NO production alone is not
sufficient for tolerance development. Chronic administration of morphine
produced a significant increase in NO production, which reduced to a moderate
level when morphine was combined with the swim stress. Therefore, large
increases in NO levels may be responsible for tolerance development and its
inhibition, even to a moderate level by the swim stress, could delay the
tolerance process.

Acknowledgement

The authors would like to thank Dr.
Fereshteh Motamadi and Miss shabnam Kharazi (Neuroscience Research Center, Shaheed Beheshti University of Medical Sciences) for their assistance in preparing the
manuscript.

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